Development of a trispecific fusion protein based on angiotensin-converting enzyme 2, glycoprotein 130, and tumor necrosis factor receptor 2 as a promising therapeutic for COVID-19.

Despite a substantial reduction in the incidence of coronavirus disease 2019 (COVID-19) infections, severe cases continue to pose a significant clinical burden, particularly among elderly individuals and patients with underlying medical conditions, due to high viral loads and cytokine storm syndrome. Elevated levels of interleukin-6 (IL-6) and tumor necrosis factor (TNF), signaling through their respective receptors, glycoprotein 130 (GP130)/interleukin-6 receptor (IL-6R) and tumor necrosis factor receptor 2 (TNFR2), are independent predictors of disease severity and mortality. To address this challenge, a series of bifunctional and trifunctional decoy receptor fusion proteins were developed by fusing the extracellular domains of TNFR2 and/or GP130 to an engineered angiotensin-converting enzyme 2 (ACE2) protein, the entry receptor for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Five mutations (T27F, K31Y, L79W, R273Q, and N330Y) were introduced into the ACE2 domain to enhance its binding affinity and neutralizing activity against a broad range of SARS-CoV-2 variants, including the currently circulating JN.1 variant. The TNFR2 and GP130 domain confer strong binding to TNF and IL-6R-IL-6 complex, respectively, thereby effectively blocking pro-inflammatory signaling pathways. In a mouse model of acute lung inflammation induced by R848, treatment with the bifunctional and trifunctional fusion proteins markedly attenuated pulmonary pathology by dampening IL-6- and TNF-mediated inflammation. These findings demonstrate a promising therapeutic strategy for severe COVID-19 and offer a framework for designing multifunctional biologics against emerging viral infections.